This invention relates to a sample loading sheet for loading an assay sample in specified lane positions in a gel electrolyte layer in an electrophoresis plate to be used in a gel electrophoretic apparatus. More particularly, the invention relates to a sample loading sheet for loading an assay sample in specified lane positions in a gel electrolyte layer in an electrophoresis plate to be used in a gel electrophoretic apparatus such as a DNA base sequencer capable of determining the base sequences of DNA by fluorescence labeling in an efficient and rapid manner.
Gel electrophoresis is practiced extensively as a technique for determining the base sequences of DNA and other proteins.
Conventionally, the sample to be subjected to electrophoresis is labelled with a radioisotope for analysis but this method has had the problem of being painstaking and time-consuming. Furthermore, the use of radioactive substances always calls for utmost safety and management and analysis cannot be performed in areas other than facilities that clear certain regulations. Under the circumstances, a method that uses fluorophores to label the sample and which detects fluorescences as emitted upon irradiation with light is being reviewed.
In this method, fluorophore-labelled DNA fragments are caused to migrate through a gel and a light excitation portion and a photodetector are provided for each electrophoresis track in an area 10-70 cm below the start point of electrophoresis. The DNA fragments are assayed as they pass through the line connecting the light excitation portion and the photodetector. A typical procedure of the method is described below. First, using as a template the DNA chain to be determined for its base sequence, DNAs of various lengths with known terminal base species are replicated by a method involving an enzymatic reaction (the dideoxy method). Then, the replicated DNAs are labelled with a fluorophore. Stated more specifically, there are prepared a group of adenine (A) fragments, a group of cytosine (C) fragments, a group of guanine (G) fragments and a group of thymine (T) fragments, all being labelled with a fluorophore. A mixture of these fragment groups is injected into separate lane grooves in an electrophoretic gel and, thereafter, a voltage is applied at opposite ends of the gel. Since DNA is a chained polymer with negative charges, it will move across the gel at a rate in inverse proportion to its molecular weight. The shorter the DNA chain (the smaller its molecular weight), the faster will it move and vice versa; this is the principle behind the fractionation of DNA by molecular weight.
Japanese Laid-Open Patent Application (kokai) No. 21556/1988 teaches a DNA base sequencer that is adapted in such a way that a line on the gel in an apparatus for electrophoresis at which laser light is applied and the direction in which photodiodes are arranged are both perpendicular to the direction in which DNA fragments migrate in the apparatus.
The setup of this apparatus is shown schematically in FIG. 14. In the apparatus shown in FIG. 14, a laser beam emitted from a light source 70 is reflected by a mirror 72 and launched horizontally from one side of an electrophoresis plate 74 at a predetermined point on the gel. As the fluorescence-labelled DNA fragments migrating through the gel pass through the irradiated region, they will fluoresce successively. The horizontal position of fluorescence emission tells the species of a particular terminal base, the time difference from the start of migration tells the length of a particular fragment, and the emission wavelength identifies the sample under assay. The fluorescence from each electrophoresis track is condensed by a lens 78 to focus at a light-receiving area 82 in an image intensifier 80. The received signal is amplified and converted to an electric signal in a photodiode array 84 for the purpose of various measurements. The results of measurements are processed with a computer so that the sequences of the individual DNA fragments are calculated to determine the base sequence of the DNA at issue.
As shown in FIG. 15, the electrophoresis plate 74 comprises a pair of glass plates 86 and 88 between which is held a gel electrolyte layer 90 made of an electrophoresing gel (e.g. polyacrylamide gel). To regulate the thickness of the gel electrolyte layer 90, a spacer 92 is provided between the two glass plates along both vertical edges. The top edge of the glass plate 88 is cut away in a specified depth across the entire width except both lateral ends. The resulting cutout 94 provides access for a buffer solution to make contact with the top edge of gel electrolyte layer 90. The electrophoresis plate 74 has an overall thickness of about 10 mm but the thickness of the gel electrolyte layer itself is only about 0.3 mm. The upper edge of the gel electrolyte layer is comb-shaped (i.e., has indentations) and located substantially flush with the bottom 96 of the cutout 94. Fluorophore-labelled DNA fragments to be assayed are injected into grooves 75 between the teeth of the comb.
Each of the grooves 75 into which the DNA fragments are to be injected has a width of about 1.5 mm and a depth of no more than about 5 mm. Two grooves are spaced apart by a distance of about 2 mm. Such small dimensions require that a fine glass tube, such as a capillary, be used to inject the samples into the grooves 75. However, due to the transparency of the glass plates and the gel electrolyte, identifying or determining the positions of the individual grooves 75 is extremely difficult and the failure to inject the samples into the right grooves has been frequent.
To support the injection of DNA samples, a sharktooth comb of the shape shown in FIG. 16 has been developed and used. The sharktooth comb is described in U.S. Pat. No. 5,744,097 which was issued to Machida et al. on Apr. 28, 1998 and herein incorporated by reference. The sharktooth comb indicated by 110 in FIG. 16 has a series of teeth 113 formed on one of its longer sides. The sharktooth comb 110 may be made of a water-swellable material such as paper. As shown in FIG. 17, the sharktooth comb 110 is inserted, usually from the top edge of the electrophoresis plate 74, into the gap between the two glass plates. The tips of the teeth 113 of the sharktooth comb 110 are slightly urged into the gel electrolyte layer. When the sharktooth comb 110 is immersed in a buffer solution, they swell and close all gaps present between the two glass plates. As a result, adjacent teeth 113 form walls that isolate two adjacent sample loading zones that are defined by spaces 115.
FIG. 18 is a section taken on line XVIIIxe2x80x94XVIII of FIG. 17. As shown, the top edge of the glass plate 88 which combines with the other glass plate 86 to form the electrophoresis plate is partly cut away in the longitudinal direction. Since the teeth 113 have a specified length, an opening 117 is formed between the root of each tooth and the bottom edge 96 of the cutout 94 in the glass plate 88. In the actual sample injecting process, the operator 13 looking through the glass plate 86 inserts the tip of a micro-injecting device 119 such as a capillary or plate chip into the sample loading space 115 via the opening 117 and injects a liquid sample 121. This process involves extreme difficulty in checking the right injecting site through the glass plate and occasionally suffers from the problem of clogging of the injection device 119. The problem of clogging can be avoided by substituting a micropipette but its use is not practically feasible since the openings 117 are very difficult to see and physically too small for the micropipette to be inserted.
To determine the base sequences of DNA, the four bases that compose the DNA, i.e., adenine (A), guanine (G), cytosine (C) and thymine (T), must be detected according to the correct order. A failure in sample injection is most likely to cause an error in the result of analysis. Hence, sample injection requires utmost care, which has been one of the reasons for the substantial drop in the operational efficiency. In particular, the failure to inject a sample into one electrophoretic track results in the need to repeat the injecting procedure over again for the whole electrophoresis plate and the mental and physical fatigue on the side of the operator who is injecting the sample is by no means negligible.
An object, therefore, of the present invention is to provide a sample loading sheet for use with a gel electrophoretic apparatus that substantially reduces the volume of operation of injecting DNA samples.
This object of the invention can be attained by a sampling loading sheet for loading an assay sample in specified lane positions in a gel electrolyte layer in an electrophoresis plate to be used in a gel electrophoretic apparatus, characterized in that said sheet is formed of cation-exchange chromatographic paper and has part or all of at least one principal surface thereof coated with a water-resistant resin film.
The sample loading sheet of the invention is formed of cation-exchange chromatographic paper. A DNA sample to be assayed is adsorbed by the sheet in specified positions. The sheet is then fitted in the electrophoresis plate in a gel electrophoretic apparatus and electrophoresis is performed under an applied electric voltage. Conventionally, the DNA sample was directly injected into the gel electrolyte layer in the electrophoresis plate but this cumbersome step is eliminated by using the sampling loading sheet of the invention. Although no exact mechanism has been unraveled, the following explanation may be put forward as a probable hypothesis: the cation-exchange chromatographic paper of which the sample loading sheet of the invention is formed has such low adsorbability of a fluorescence-labeled DNA sample that upon application of an electrophoretic voltage, the sample is detached from the sheet at a sufficiently high speed that the resolution is almost comparable to what is obtained by the conventional procedure of electrophoresis in which the fluorescence-labeled DNA sample is directly injected into the gel electrolyte layer.